Analysis of carrier ampholytes using immobilized ph gradients

ABSTRACT

New synthesis and analysis procedures for novel crosslinked polyamines and ampholytes. Polyamines are crosslinked with tartaric acid ester, malonic acid ester, or polycarboxylic acid esters of the citric acid cycle. The resulting crosslinked polyamine may further react with other compounds such as an α, β-unsaturated or α-halo unsaturated carboxylic acids to prepare new ampholyte mixtures. The resulting novel ampholytes exhibit greater heterogeneity and complexity than presently prepared ampholytes, and can be used in analytical and preparative isoelectric focusing processes. 
     Novel ampholyte analysis process entails analyzing chemical compounds, usually ampholytes, used in isoelectric focusing processes. The ampholyte is isoelectrically focused on an immobilized pH gradient, and then immersed in a picric acid solution to c 
     GOVERNMENT OWNERSHIP 
     The funding for the subject matter described herein was partially provided by the U.S. Government under Grant Nos. NAS 9-17403 and NIH AI-20590. The U.S. Government may retain certain royalty-free rights in the invention claimed herein.

GOVERNMENT OWNERSHIP

The funding for the subject matter described herein was partiallyprovided by the U.S. Government under Grant Nos. NAS 9-17403 and NIHAI-20590. The U.S. Government may retain certain royalty-free rights inthe invention claimed herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is concerned with a new synthesis procedure forpreparing novel crosslinked polyamines. The crosslinked polyamines maybe used to synthesize new zwitterionic compounds suitable for use asampholytes in isoelectric focusing of amphoteric substances.

In addition, this invention relates generally to the field of chemicalcompound analysis. More specifically, a method is described for analysisof carrier ampholytes used in isoelectric focusing. The method includesfocusing carrier ampholytes on immobilized ph gradients and thenvisualizing the ampholytes by precipitation using precipitatingsolutions such as picric acid.

2. Description of the Related Art

Ampholyte Synthesis

Isoelectric focusing ("IEF") is a known process for resolving individualmolecular species under either denaturing or non-denaturing conditions.IEF is used to separate, purify, and analyze amphoteric substances suchas proteins, enzymes, hormones, antigens, antibodies, etc. IEF is aprocess wherein an applied electrical field forces heterogeneous carrierzwitterions, zwitterionic mixtures, or ampholytes to segregate into anordered array of molecules, thus establishing a pH gradient between theanode and cathode electrodes. This procedure can be carried out ineither a free solution format or in a gel format.

A zwitterion is a molecule that has at least one region of positivecharge and at least one region of negative charge. Ampholytes aresubstances that may ionize to form either anions or cations. Ampholytesmay comprise zwitterions and vice versa. Preferentially, ampholytes usedin this invention comprise a mixture of numerous species of zwitterionicchemicals which differ from each other by the nature and number of basicand acidic groups.

Each ampholyte species in an ampholyte mixture has its own intrinsicisoelectric point. In a particularly suitable system of carrierampholytes, the isoelectric points of the different ampholyte specieswill cover the pH range 3-10. Most naturally occurring proteins,enzymes, hormones, antigens, antibodies, etc. are isoelectric at somepoint within this pH range. Substantially uniform distribution of theinherent isoelectric points of the various ampholyte species throughoutthe desired pH range is an important factor for the formation of uniformand linear pH gradients.

U.S. Pat. Nos. 4,131,534 to Just, 3,901,780 to Denckla, 3,692,654 toSvendsen, and 3,485,736 to Vesterberg all outline various methods forpreparing carrier ampholytes.

One well-known method of preparing carrier ampholytes involvessynthesizing polyamine-polycarboxylic acid mixtures for use as carrierampholytes. This method typically utilizes a polyethylene polyamine towhich an α,β-unsaturated acid such as acrylic acid is chemically linkedby an addition reaction. The simplest ampholyte formed by that reactionis a polyethylene polyamine molecule to which one carboxylic acidmolecule is linked, forming a β-amino carboxylic acid system. Becausemultiple amino groups are present in the molecule, the addition reactioncan occur more than once. Subsequent addition reactions yield β-aminopolycarboxylic acids which contain an increasing number of carboxylicacids in the molecule.

As a result of the above synthesis, some of the resulting ampholytespecies are obtained in a high yield whereas other species will beformed at the same time in relatively low yield. A suitable ampholytemixture should preferably exhibit a uniform distribution of bufferingcapacity and conductivity throughout the pH gradient and preferablyprovide a large number of buffering species per pH unit. The ampholytemixture should preferably be evenly distributed across the pH span. Thepreferable conditions may be achieved when the different ampholytespecies are contained in the mixture in approximately equalconcentrations. Therefore, at the end of the chemical reaction,fractionation of the ampholyte mixture in expensive multicompartmentelectrolysis equipment is normally necessary to blend acceptablemixtures by boosting the concentrations of species obtained at low yieldin the initial synthesis. A useful ampholyte mixture is then obtained bymixing appropriate amounts of materials from different electrolysiscompartments.

The above method of synthesis makes use of known organic chemistryprocedures. There are numerous examples in the literature in whichnucleophilic substances (such as the amino groups here) react withelectron-deficient alkenes (i.e. acrylic acid) to afford additionproducts. In these instances, it has been demonstrated that the additionreaction yields favorable results when equimolar quantities of eachreactant are utilized.

The above method of synthesis involves adding a calibrated amount ofaqueous unsaturated carboxylic acid to an aqueous solution ofpolyethylene polyamine, with simultaneous heating and stirring. Insteadof a single amine, amine mixtures may be used. Similarly, instead of asingle carboxylic acid, a mixture of several carboxylic acids eachcontaining at least one carbon-carbon double bond within their moleculesmay be utilized.

Since the reaction between the unsaturated carboxylic acid and thepolyethylene polyamine exhibits slow reaction kinetics at roomtemperature, the reaction is generally performed at elevatedtemperature. This procedure, however, leads to undesired side productswhich cause coloration of the product. Additionally, even at elevatedtemperature, the reaction kinetics are rather slow and it takes severalhours for all of the unsaturated carboxylic acid to react.

A problem with the prior art synthesis of carrier ampholyte mixtures isthat the starting polyethylene polyamine compounds which arecommercially available come in only a limited number of straight chainforms. Thus, the number of different molecular species or isomers ofampholytes that can be made from these is quite limited. Because of allof the above problems, known procedures are not completely satisfactory,and persons skilled in the art have searched for improvements.

Ampholyte Analysis

The resolving power of IEF is largely dependent upon the nature of thepH gradient used. Ideally, the pH gradient should be stepless with aneven distribution of charged groups throughout the pH range of interest.Immobilized pH gradients ("IPGs") have overcome many of the problemsassociated with the uneven distribution of charged groups. However, IPGsare generally limited to analytical and small scale preparativeprocedures using polyacrylamide matrices. In certain applications IPGanalysis requires carrier ampholytes to avoid protein-matrixinteractions.

In contrast to IPGs, carrier ampholytes ("CA") have the advantage ofbeing used with virtually any IEF support medium (polyacrylamide,agarose, Sephadex, etc.) or with liquid phase preparative equipment. Theresolution of a CA gradient is dependent upon the number and quality ofthe ampholyte species which are used to generate it. The distribution ofampholyte species over a given pH range becomes especially importantwhen considering narrow range preparative or analytical procedures. Forresearch efforts which are aimed at producing more diverse ampholytes itis preferable to have a reproducible method for monitoring theapproximate number and linear charge distribution of the ampholytespecies which have been synthesized.

Attempts to estimate the total number of molecular species in ampholytepreparations have met with criticism. Some practitioners have emphasizedthat the absolute number of molecular species in an ampholytepreparation is of limited importance as compared to the bufferingcapacity of the species which are present. Nevertheless, such methodshave been helpful for evaluating the relative heterogeneity of ampholytespecies.

One method of estimating the total number of molecular species involvesfocusing ampholytes in Sephadex (Pharmacia-LKB Biotechnology [LKB],Bromma, Sweden), then rolling a sheet of filter paper saturated with 5%glucose onto the gel surface. The filter paper is then removed andheated at 110° C. to produce visible ampholyte-glucose caramel reactionproducts. Using this technique, it was estimated that LKB wide rangeAmpholines contain approximately 62 carrier species. In another method,a paper print of focused ampholytes is treated with formaldehyde,lactose, or ninhydrin. Using this method, wide range LKB Ampholines havebeen estimated to comprise more than 500 individual species.Discontinuities in ampholyte distribution have been detected bySchlieren patterns and side illumination of focused gels.

Carrier ampholyte heterogeneity has also been studied using methodswhich attempted to estimate the distribution of conductivity, and theability of ampholyte preparations to resolve focused proteins. Althoughthe last approach may be the most direct for choosing an ampholytemixture for separation of specific groups of proteins, its broadapplication is limited by the lack of a suitable protein preparationwhich contains a continuum of closely spaced, well-defined proteinspecies.

All of the above-mentioned analytical methods rely on conventional IEFin gradients which are generated by the ampholytes themselves. Incontrast, IPG analysis separates ampholytes in a gradient which isdictated by a smooth and continuous distribution of charged groupscovalently bound on a gel matrix. Individual ampholyte species migrateto a position in the pH continuum of the IPG which is indicative oftheir pI.

Prior to this invention, no method known to the inventors has beendeveloped to analyze ampholytes using a constant calibrated medium tocompare the heterogeneity of different ampholyte compounds.

SUMMARY OF THE INVENTION Ampholyte Synthesis

A general object of this invention is to make more complex andheterogeneous polyamine mixtures. These mixtures can then be used toprepare ampholytes of greater complexity and therefore, greaterresolution. The ampholytes of this invention may be used for eitheranalytical or preparative purposes.

One advantage of using crosslinked polyamines for making ampholytes isthat the resulting ampholytes may be fractionated to prepare narrow pHgradients. Because the novel ampholytes are more heterogeneous andcomplex than previous ampholytes, better resolution of molecules thathave similar isoelectric points is possible.

One reason that commercially available ampholytes lack heterogeneity isthat the commercially available polyamine starting materials areavailable in only a limited number of straight chain forms [e.g.,diethylenetriamine (DETA), triethylenetetramine (TETA),tetraethylenepentamine (TEPA), and pentaethylenehexamine (PEHA)]. Thusthe number of ampholyte species created is correspondingly limited. Thepresent invention approaches this problem by crosslinking polyamines togenerate larger polyamines with highly complex isomeric forms.

An advantage of the present invention is that the crosslinking compoundsdo not add ionically charged groups to the polyamines during thereaction. Adding ionically charged groups to the polyamines could bedetrimental to subsequent synthesis of ampholytes, since preferablyampholytes are synthesized in a basic pH environment.

Another advantage of this invention is that the reactions proceedquickly and efficiently.

Generally, a first aspect of this invention includes preparing acrosslinked polyamine compound, comprising the reaction product of apolyamine and a crosslinking reactant, wherein the crosslinking reactantis a tartaric acid ester (such as diethyltartaric acid ester ("diethyltartrate")), a malonic acid ester, a polycarboxylic acid ester of thecitric acid cycle, or mixtures thereof. A second aspect of thisinvention includes preparing an ampholyte mixture, comprising thereaction product of a crosslinked polyamine compound and anα,β-unsaturated carboxylic acid such as acrylic acid.

Ampholyte Analysis

Once an ampholyte mixture is prepared, it is desirable to analyze it todetermine its heterogeneity. The ampholyte analysis aspect of thisinvention uses IPGs for the separation and subsequent analysis ofampholyte heterogeneity and distribution. Under these conditions the pHgradient is dictated by the immobilized groups. Typically, a gelcontaining the immobilized pH gradient is washed, prefocused, and cutinto strips prior to application of an ampholyte to the gradient. Uponapplication, the ampholyte is itself focused to separate the variousampholyte species. The individual ampholyte species, during IEF, migrateto their individual isoelectric points. The gel is then immersed in asolution containing chemicals which cause in situ precipitation of theseparated ampholyte species. A saturated solution of picric acid ispreferably used to precipitate the ampholyte species so that they can bedetected. The different species appear as distinct bands, each separateband indicating the presence of at least one distinct ampholyte species.The bands may be photographed for comparative or archival purposes orthey may be scanned in a densitometer for quantitation of the relativecontent of ampholytes found in individual bands.

Using this invention, significant variations between differentcommercial sources of ampholytes, as well as ampholytes synthesized inthe laboratory, are apparent. An ampholyte that contains fewer molecularspecies exhibits only a limited number of bands and an ampholytecontaining a large number of molecular species exhibits numerous bands.Thus, one advantage of this invention is that it provides a relativelyquick, simple, and inexpensive method of analyzing chemical compoundheterogeneity, especially ampholyte heterogeneity.

Another advantage of this invention is that a reproducible immobilizedpH gradient system is used to analyze the ampholytes. The pH gradient isimmobile and reproducible because the chemicals which generate thegradient may be obtained in a highly purified state, may be accuratelyweighed, and are coupled covalently to the acrylamide gel matrix duringpolymerization of the gel. Thus, the position in the gel where theindividual ampholyte molecules migrate in an electrical field is notdictated by the ampholyte mixture itself, but instead is dictated by thecommercially available and reproducible immobilized pH gradient. Ineffect, this invention provides a method to create an independent pHgradient for separation and eventual visual or quantitative analysis ofampholytes.

This invention may be used to help create ampholytes with improvedproperties. As is shown in the preferred embodiment, a relationshipexists between ampholyte heterogeneity as demonstrated by this inventionand the resolution of proteins in IPGs. Since the immobilized pHgradient is constant and reproducible, testing of different ampholytecompounds using this invention enables practitioners to compareampholytes. Thus, a practitioner can analyze two different ampholytes onthe same immobilized pH gradient and compare them to one another (or toa previous standard) for the degree of heterogeneity. Therefore,commercially available immobilized pH gradients may be used as a"standard" for testing ampholytes.

A practitioner may also use the invention described in this disclosureto select and test synthesized and commercial ampholytes for specificapplications. For instance, using this invention a practitioner may beable to test and select an ampholyte that exhibits greater resolution inone narrow pH range and a second ampholyte that exhibits greaterresolution in a different narrow pH range.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a photograph of several synthesized TETA and TEPA derivativeampholytes visualized on immobilized pH gradients.

FIG. 2 is a photograph of several synthesized and several commercialampholytes visualized on immobilized pH gradients.

FIG. 3 is a photograph of the results of IPG picric acid analysis ofseveral commercial ampholytes.

FIG. 4 is a photograph of the results of IPG picric acid analysis ofseveral synthetic and commercial ampholytes.

FIGS. 5A and B are photographs of the results of conventional IEF ofserum proteins using ampholytes which were synthesized withtetraethylenepentamine ("TEPA") or pentaethylenehexamine ("PEHA"), andacrylic acid.

FIG. 6 is a photograph of the results of IPG picric acid analysis ofampholytes synthesized with bis-acrylamide crosslinked polyamines.

DESCRIPTION OF PREFERRED EMBODIMENTS Ampholyte Synthesis

A first aspect of this invention comprises crosslinking polyamines withreactants such as tartaric acid ester, malonic acid ester, or carboxylicacid esters of the citric acid cycle. These acid esters may be in the L,D, meso, or racemic form. For instance, L-diethyltartaric acid ester,D-diethyltartaric acid ester, meso-diethyltartaric acid ester, orracemic diethyltartaric acid ester may be used. In addition, the alkylportion of the esters may be replaced with other alkyl radicals (otherthan ethyl) such as methyl, propyl, or isopropyl radicals. The esters,when added to a polyamine compound in the presence of heat withoutoxygen, crosslink the polyamine compounds, creating a large number ofheterogenous polyamine crosslinked isomers.

In a second aspect of the invention the crosslinked polyamine compoundsare reacted with an unsaturated carboxylic acid to form heterogenousampholyte mixtures. Thus, crosslinked polyamines may be reacted with anα,β-unsaturated carboxylic acid such as acrylic acid to form anampholyte mixture. Other suitable α,β-unsaturated carboxylic acidscomprise methacrylic acid, methylene malonic acid, ethylene malonicacid, crotonic acid, maleic acid, fumaric acid, itaconic acid, ormixtures thereof. The resulting ampholyte may be diluted with water andcooled, or otherwise allowed to cool, to ambient temperatures. Samplesof the resulting ampholytes may be isoelectrically focused on an IPG,precipitated, and photographed for analytical purposes. The ampholytemay be subsequently used as a carrier ampholyte to separate amphotericsubstances such as proteins, enzymes, antibodies, antigens, hormones,etc.

In all reactions contemplated, polyamine mixtures may be used instead ofa single polyamine. Furthermore, a mixture of crosslinking reactantscomprising tartaric acid ester, malonic acid ester, or a polycarboxylicacid ester of the citric acid cycle may be utilized. Mixtures of any ofthe reactants in the subject invention may be used to provide for a moreheterogenous ampholyte mixture.

A particular advantage of the present invention is that it may usecommercially available polyamines, crosslinking reactants, andα,β-unsaturated carboxylic acids. All of these compounds are relativelyinexpensive and are available in an acceptable purity grade.

Polyamine compounds which are employed for the synthesis of carrierampholytes will in most embodiments contain at least two amino or iminogroups. Most polyamines which are suitable for the present procedurewill comprise the following unit: --NH--R-- NH--. In addition, it isanticipated that some polyamines may also comprise peptides.

The chain length (--NH--R--NH--) of the polyamine may be prolonged inboth directions with similar --R--NH-- units. For special purposes,resulting compounds may have molecular weights of 20,000 or more. In theabove formula, R may be --C₃ H₆ --, --CH₂ --, or preferably --C₂ H₄ --.Typically, the organic nitrogen compounds will be linear chainmolecules. Moreover, various nitrogen atoms may be substituted by alkylgroups which may contain 1-6 carbon atoms. Preferred polyamines arepolyethylene polyamines such as DETA, TETA, TEPA, and PEHA.

In addition to the above polyamines, it is contemplated that otherpolyamines such as peptides may be used. It is envisioned that mostuseful peptides will comprise at least two amino groups and a chain of3-20 amino acids, preferably 3-10 amino acids. A chain length of 3-10amino acids is preferred due to the relatively low molecular weight ofthe final product. The lower molecular weight generally makes it easierto separate the resulting ampholyte from the larger amphoteric proteinmolecules after IEF. Examples of peptides that are contemplated to beuseful as polyamines include peptides comprising basic amino acids suchas L-lysine, D-lysine, L-arginine, D-arginine, L-histidine, D-histidine,or mixtures thereof. In addition, mixtures of different peptidescomprised of different sequences of amino acids are also contemplated.Thus, one peptide comprised of a particular sequence of amino acids maybe mixed with one or more peptides comprised of different sequences ofamino acids. Varying chain length peptides may also be mixed.

Peptide polyamines outlined above may be mixed with other polyaminessuch as polyethylene polyamines to achieve a more heterogeneous mixtureof starting polyamine. The more complex and heterogeneous the startingpolyamine, the more likely the resulting ampholyte mixture will becomplex and heterogeneous, thus increasing the resolution potential ofan ampholyte mixture.

The preferred crosslinking reactants comprise tartaric acid ester,malonic acid ester, and carboxylic acid esters of the citric acid cycle.The citric acid cycle is a well-known biological chemical reactionsequence, and it is contemplated that suitable carboxylic acid estersfrom that cycle include maleic acid ester, oxaloacetic acid ester,citric acid ester, aconitic acid ester, isocitric acid ester,oxalosuccinic acid ester, and α-ketoglutaric acid ester.

Hydroxyl groups present in most of the above-named esters impart twoproperties to the resulting crosslinked polyamine. First, the presenceof hydroxyl groups increases the water solubility of the crosslinkedpolyamine by making it more hydrophilic. Second, the hydroxyl groupsserve as hydrogen-bonding acceptors and donors. Most biologicalmolecules (i.e. enzymes, proteins, antibodies, antigens, etc.) dependheavily upon hydrogen-bonding for the maintenance of theirthree-dimensional integrity. The ability of the crosslinked polyaminesto participate in hydrogen-bonding generally results in increasedinteraction between the molecules to be purified or analyzed, and theampholytes prepared from the crosslinked polyamines. Thus, the resultingampholytes, because of the hydrogen-bonding properties of thecrosslinking reactants, exhibit improved separations.

In one embodiment, the unsaturated acid monomers which are used in theinvention contain a carbon-carbon double bond activated for addition tothe amino group. Carboxylic acids with a double bond in the α,β-positionare preferably used although the double bond may be positioned elsewherewithin the molecule and the molecule may be activated otherwise.

For the controlled synthesis of an ampholyte mixture, gradual mixing ofthe starting materials is employed. Heating the reactants in anoxygen-free atmosphere such as an inert gas (nitrogen) is part of thesynthesis process. The mixing ratios of the two reactants may becalculated with accuracy from the molecular weights of the reactants.For example, since each acrylic acid molecule has one amino-reactivegroup and each PEHA molecule has two primary amino groups and foursecondary amino groups, it is predictable that even at total saturationonly a maximum of eight acrylic acid molecules could couple to each PEHAmolecule. Obviously, any number less than eight acrylic acid moleculescould couple to each molecule of PEHA on average.

In practice, approximately one mole of α,β-unsaturated acid is reactedfor every two available primary or secondary amino groups to make anacceptable ampholyte with a pH range from 3-10. Thus, for example, forevery mole of PEHA, which has six available amino groups, three moles ofacrylic acid is required to react with the PEHA to make a pH 3-10ampholyte mixture. "Available" amino groups should be interpreted tomean primary or secondary amino groups. If two moles of PEHA arecrosslinked with diethyltartaric acid ester, then only ten amino groupsare available and therefore five moles of acrylic acid are theoreticallyrequired.

The present procedure produces a carrier ampholyte mixture which mayhave a slight yellow-brown coloration. To remove that coloration and tolower absorption in the ultraviolet region of the spectrum, the carrierampholyte mixture may be treated with hydrogen in the presence of asuitable catalyst. Raney-nickel, platinum oxide, palladium,palladium-activated carbon, or hydrides such as lithium aluminum hydrideand sodium borohydride may be used. The hydrogenation is preferablyperformed in aqueous solution but may also be carried out in a suitableorganic solvent. In addition, activated charcoal chromatography may alsobe used for decolorization.

One preferred embodiment was prepared as follows. A crosslinkedpolyamine compound was prepared by adding diethyltartaric acid esterwith a polyamine in a tetrahydrofuran solvent solution. Other solventsthat may work acceptably in this application include ether, dioxane andbenzene. The diethyltartaric acid ester was added dropwise whilestirring the mixture. The diethyltartaric acid ester and the polyaminewere added in substantially equal molar quantities and heated at refluxfor approximately 6 hours. The atmosphere of the reaction vessel wasflushed with anhydrous oxygen-free gas (nitrogen gas), and the vesselwas sealed and heated at approximately 50° C. for approximately 12-16hours. Methane, any other hydrocarbon gas, or any of the inert noblegases (helium, neon, argon, krypton, xenon) may also be used as anoxygen-free atmosphere for this invention. Following heating, thetetrahydrofuran solvent was removed by evaporation in a partial vacuumof around 30 mm Hg. The result was a complexed crosslinked polyaminecompound.

Following synthesis of the crosslinked polyamine compound, that compoundmay be stored or used to synthesize an ampholyte. If an ampholyte is tobe prepared, the crosslinked polyamine compound is diluted with waterand mixed with an appropriate amount of α,β-unsaturated carboxylic acid.It is well understood to persons skilled in the art that water may beadded in varying amounts to achieve desired compound concentrations. Theα,β-unsaturated carboxylic acid used in one preferred embodiment wasacrylic acid and the polyamine compound was first cooled to atemperature of about 25°-30° C.

After adding acrylic acid in the above examples, the atmosphere of thevessel was once again flushed with an oxygen-free atmosphere and thevessel was then sealed and heated to approximately 70° C., withstirring, for approximately 16 hours. Water was then added to bring theampholyte solution to a final concentration of 40% (weight/volume). Thisis a generally appropriate concentration although any otherconcentration is acceptable.

The polyamine compound may be a mixture of various polyamines. Inaddition, the relative amount of tartaric acid ester may be varied. Inone preferred embodiment, a molar ratio of 3:4 (tartaric acidester:polyamine) was used. Similarly, the molar ratios of availablepolyamine amino groups to unsaturated α,β-unsaturated carboxylic acidmay also be varied. A molar ratio of 2:1 (available polyamineamino:α,β-unsaturated carboxylic acid) yielded optimal ampholytes withthe mixtures utilized. It will be understood by those skilled in the artthat the molar ratios may be varied to prepare different complexedpolyamine mixtures.

Similarly, it will also be understood that the temperatures and times ofthe reactions of the invention may also be varied. It is contemplated,for example, that the crosslinking reaction may take place within atemperature range of 30√-140° C., preferably 40°-60° C. and for a periodof 2-48 hours, preferably 12-20 hours. When an unsaturated carboxylicacid is added to the crosslinked reactant, that reaction may take placeat a temperature range of 50°-140° C., preferably 60°-80° C., and a timeperiod of 2-48 hours, preferably 12-20 hours. It is anticipated thathigher temperatures will normally require less reaction time and viceversa.

TETA, TEPA, and PEHA were all crosslinked in molar ratios of 4:1, 4:2,4:3, 4:4, and 4:5 of polyamine to L-diethyltartaric acid ester. A 2:1ratio of available crosslinked polyamine amino groups to acrylic acidwas subsequently utilized to synthesize ampholytes. The ampholytes werethen isoelectrically focused and analyzed using commercial solid statepH gradient media (LKB). The analysis was completed according to theprocess described below in the "Ampholyte Analysis" section herein. Forcomparative purposes, ampholytes synthesized from TETA, TEPA, and PEHAmonomers were also visualized, along with commercial ampholytes such asATI (Ampholife Technologies, Inc., The Woodlands, TX) and LKBampholytes. FIGS. 1 and 2 show the analysis of ampholytes using themethods described. Visual inspection of the patterns is a qualitativemethod of comparing the ampholytes and provides a rapid method ofanalysis to assess success or failure in modifying protocols forampholyte synthesis, but is not necessary to create improved ampholytesas described herein. When quantitative results are desired, thedistribution of precipitated bands in the gels can be analyzed usingdensitometric measurement. One densitometer that may be used is the"Quick Scan Jr." made by Helena Laboratories (Beaumont, Texas).

The number of regions (in this case, bands) of precipitate seen on thegels gives a minimum estimate of the number of isoelectrically focusedampholyte species. In general, larger numbers of bands in the gelcorrespond to more heterogeneous and complex ampholytes (a favoredresult).

As is readily apparent from FIGS. 1 and 2, the ampholytes that weresynthesized using the crosslinking reactant (tartaric acid ester) weresignificantly more heterogenous and complex than the ampholytes madefrom TETA, TEPA, and PEHA monomers. In addition, some of the PEHAampholytes that were crosslinked with tartaric acid ester exhibitedgreater heterogeneity and complexity than commercially available ATI andLKB ampholytes. Since some of the new ampholytes are more heterogeneousand complex than existing commercially available ampholytes, it isenvisioned that still other new ampholytes will achieve correspondinglyhigher resolutions. For instance, different polyamine starting materialssuch as peptides or peptidepolyethylene polyamine mixtures arecontemplated. In like manner, alternate crosslinking reactants (some ofwhich are identified herein) may be utilized.

In addition to having a greater number of ampholyte species, many of theampholytes made by using crosslinked polyamines are more evenlydistributed over the pH range and lack the dominant species which weredetected in some preparations. These dominant species may contribute touneven conductivity when the ampholytes are used for conventional IEF.

It is envisioned that all or portions (corresponding to narrower pHranges) of the new ampholytes may be used to achieve improvedseparations of amphoteric substances. In addition, the process ofcrosslinking polyamines as described in this invention will serve as auseful method for making other novel improved ampholytes. Thus, otherampholyte synthesis processes such as already described in the art maybe combined with the crosslinking procedures of this invention toprepare improved ampholytes.

Ampholyte Analysis

a. Ampholytes

Commercial ampholytes were obtained from Bio--Rad (Biolytes, pH 3-10),ICN (Iso-Lytes, pH 3-10), Geltech (Technolytes, pH 3.5-9.5) and LKB(Ampholines, pH 3.5-9.5). Ampholytes 1-8, 1A, 2A and 2B as shown inFIGS. 3, 4, and 6 were obtained from ATI. TETA, TEPA, and PEHAampholytes were synthesized as described by Binion and Rodkey inElectrophoresis 3, 284 (1982) using a 2:1 ratio of available polyaminenitrogen to acrylic acid. A group of ampholytes termed "UT WIDE"ampholytes were prepared by mixing ampholytes made with TEPA and PEHAmonomers.

For production of ampholytes using bis-acrylamide crosslinked polyamine,TETA or TEPA were combined with N,N'-methylene bis-acrylamide ("bis") atvarying molar ratios and mixed at room temperature overnight. Ampholyteswere then synthesized using acrylic acid as previously described (seethe well-known method outlined in the "Description of the Prior Art"section). The ampholytes described herein are illustrative examples andin no way limit the scope of the invention. Other chemical compounds orcompositions may also be analyzed using this invention, includingchemical compounds that are not ampholytes. It is understood that"chemical compound" as interpreted within the context of the claims maybe interpreted to mean one chemical compound or a mixture of chemicalcompounds. The chemical compounds should generally have a pI within thepH range of the immobilized pH gradient. The compounds analyzed shouldalso precipitate, to some degree, when contacted with a chemicalprecipitating solution (such as picric acid) after IEF on IPGs.

Of course, other ampholytes will also be used in this invention. Forexample, ampholytes may be preferentially prepared according to theprocedures outlined herein (see sections entitled "AmpholyteSynthesis").

b. IPG Analysis of Ampholytes

Polyacrylamide gels (25×12×0.5 cm) comprising 4.9% T, 3% C, andImmobiline chemicals (LKB) were used for the production of pH 4-10gradients according to the gel formulations described by Fawcett andChrambach. See Prot. Biol. Fluids, 33, 439 (1985). It is anticipatedthat gels comprising a total acrylamide concentration ("T") of 1-10%(3-7% preferred), and a percentage crosslinker of T ("C") of 1-5% (2-4%preferred) may be used for this invention. In general, the higher thepercentage of C, the tighter the gels are bound together.

In general, the IPGs comprise acrylamide derivatives, with the formulaCH₂ ═CH--CO--NH--R, where R denotes one of several possible ionizablegroups, with pK's in the pH 3.6-9.3 range. In general R may be2-acrylamido-2-methylpropane, 2-acrylamido glycolic acid, N-acryloylglycine, 3-acrylamido propanoic acid, 4-acrylamido butyric acid,2-morpholino ethylacrylamide, 3-morpholino propylacrylamide,N,N-dimethyl aminoethyl acrylamide, N,N-dimethyl aminopropylacrylamide,N,N-diethyaminopropylacrylamide, QAE-acrylamide (quaternary aminoethylacrylamide), or mixtures thereof.

IPGs are generally prepared by using nonamphoteric weak acids and basescoupled to an acrylamide backbone, mixed in such ratios as to define anydesired pH interval. The weak acids and bases are incorporated as agradient of charges (and thus immobilized) into the supportingpolyacrylamide matrix using a commercially available gradient makingdevice. The formula of IPG compounds and their synthetic routes arereported in "Synthesis of Buffers for Generating Immobilized pHgradients. I: Acidic acrylamido buffers," Applied and TheoreticalElectrophoresis 1, 99-102 (1989) and "Synthesis of Buffers forGenerating Immobilized pH gradients. II: Basic Acrylamido Buffers,"Applied and Theoretical Electrophoresis 1, 103-108 (1989). It isenvisioned that this invention will be applicable to most types of IPGs,and not limited to the IPGs described herein. For instance, discovery ofadditional chemicals which would extend the resulting IPG acid rangelower or the IPG basic range higher than the IPG chemicals describedabove would also be applicable IPG examples as would the discovery ofchemicals which could replace the chemicals described above in theirrespective pI ranges. Commercial brand LKB chemicals as described abovewere preferred.

In this embodiment, the gels were polymerized, washed in distilledwater, washed with 1% glycerol, and dried for storage. Before running,the dried gels were hydrated for 2 h in distilled water and prefocusedovernight at 0.5 W limiting power at 8° C. to remove any residualcatalysts remaining from the acrylamide polymerization (ammoniumpersulfate and tetramethylethylenediamine) and to exude excess liquidfrom the gel prior to application of the ampholyte samples. Thefollowing morning prefocusing was continued for at least 1 h limited at2.0 W and 5000 V during which time excess fluid was blotted from the gelsurface. After prefocusing, 0.5 cm lanes were cut in the gel withapproximately 0.2 cm spaces between the lanes. Ampholyte samples wereapplied to each lane (5 microliters of 4% ampholyte per lane) positionedat approximately 1 cm from the cathode. Focusing was performed for 6 hlimited at 5000 V, 0.5 mA and 2.3 W.

Upon completion of the run the gel was immediately immersed in saturatedpicric acid in water to precipitate the focused ampholytes renderingthem visible. "Visualization" is the process whereby the separatedspecies of the analyzed compound become visible on the IPG. The totalIPG pH gradient was measured using a flat surface pH electrode on aseparate lane of the same gel to which wide range LKB ampholytes hadbeen applied. This lane was removed from the gel for pH analysis beforetreatment of the remaining gel with picric acid. The resultingvisualized ampholytes were aligned and photographed as shown in FIGS.3-6.

c. Results of IPG Analysis of Commercial and Prepared Ampholytes

In FIG. 3 four commercially available wide range ampholytes and twosynthetic preparations were analyzed. Regions (bands) in the pH gradientrepresent focused ampholyte species which were precipitated in situ withpicric acid. All commercially available ampholytes tested (Described inPreferred Embodiment, Section 2a) contained picric acid precipitableampholyte species across the entire pH range. Therefore, thecharacteristics of the ampholyte species which render it precipitablewith picric acid do not appear to be dependent on the pI of theampholyte molecule.

IPG picric acid analysis of different synthetic ampholytes andcommercial ampholytes (Described in Preferred Embodiment, Section 2a) isshown in FIG. 4. The relative heterogeneity of ampholytes which weresynthesized using the polyamine PEHA as a backbone was compared toampholytes which were synthesized using the polyamines TETA or TEPA as abackbone. The TETA and TEPA patterns consist of relatively fewprecipitable species, and contain obvious deficiencies in certain areasof the gradient.

PEHA ampholytes produced a more heterogeneous pattern of precipitablespecies. Dominant species which were present in the TETA and TEPAsamples were not detected in the PEHA sample. Furthermore, gaps in thedistribution of bands which were detected in the TETA and TEPApreparations were "filled in" in the PEHA sample, providing a more evendistribution of ampholytes. Thus, a relationship appears to existbetween the relative heterogeneity of picric acid precipitable ampholytespecies and theoretically predicted heterogeneity (based on molecularsize and the theoretical number of ampholyte species which may begenerated). As would be theoretically predicted, ampholytes made fromPEHA exhibited greater ampholyte heterogeneity because of the largernumber of ampholyte species generated. In addition, any branching of thepolyamine, which is more likely with larger polyamines such as PEHA,tends to increase ampholyte heterogeneity.

The method of this invention may be valuable in monitoring theheterogeneity of ampholyte preparations. FIG. 4 shows IPG picric acidanalysis patterns of ampholytes obtained wherein the ampholytes wereproduced using the well-known synthetic procedure previously described.This procedure synthesized ampholytes using unmodified PEHA, TEPA orTETA with one mole acrylic acid for every 2 moles of available polyamineamino group. The FIG. 4 analyses demonstrate that with appropriatemanipulation ampholytes may be prepared that have greater diversity thanampholytes prepared with unmodified TETA, TEPA, or PEHA monomers.

The demonstration of a larger number of ampholyte species detected usingthe polyamine crosslinking method demonstrate that it is possible tosynthesize ampholytes which are superior with regard to isoelectricheterogeneity than presently available ampholytes. The larger number ofavailable polyamine isomers available for coupling to the acid moleculesincreases the overall heterogeneity of the resulting ampholytes. The newampholytes may have the potential for higher resolution of proteins thanless heterogeneous preparations because the larger number of carrierampholytes per linear distance unit on the IPG will provide a betterenvironment for two protein molecules with very closely-spaced pI to bedetected as two distinct bands rather than as one thick band.

d. Relationship of IPG Pattern to Protein Resolution

In designing new ampholyte synthesis procedures, it is important toconfirm that the banding pattern obtained by IPG picric acid analysis isindicative of the potential of these ampholytes to resolve proteins whenused in conventional IEF. The relationship between ampholyteheterogeneity and resolution of focused proteins is demonstrated in FIG.5. Serum proteins taken from rabbits during the course of activeimmunizations were separated by IEF using standard TEPA or PEHAampholytes made using unmodified TEPA or PEHA for ampholyte synthesiswith acrylic acid added at a ratio of one mole of acrylic acid/2 molesof available polyamine amino groups.

Flatbed IEF was performed in LKB Multiphor chambers at 8° C. to analyzethe serum proteins. Polyacrylamide gels (25×12×0.5 cm) comprised 5.3% T,3% C, and 3% ampholyte. Electrode wicks were saturated with 1 M NaOH(cathode) and 1 M H₃ PO₄ (anode). Ten microliter samples of serumdiluted 1:5 were applied to the gel surface using silicone rubbersurface applicators. The IEF protocol began with an initial setting of100 V (constant voltage) for 15 minutes, and was then raised to 200 V(constant voltage) for an additional 15 minutes. The setting was thenswitched to 5 W (constant power) for 60 minutes followed by 7 W(constant power) for an additional 30 minutes. The pH gradient wasmeasured immediately following electrophoresis using a flat surface pHelectrode. Proteins were fixed (immobilized and preserved) in the gel byplacing the gel in a fixative solution consisting of 3.45%sulfosalicylic acid, 11.5% trichloracetic acid in 30% methanol for 1 hwith rocking. The gels were then rinsed in 25% ethanol, 8% acetic acidfor 1 h, the rinse solution was changed, and a second rinse was doneovernight. The gel was then immersed in the same solution containing0.1% Coomassie Brilliant Blue R250 for 1 h to stain proteins which hadbeen previously fixed so that they could be seen with the naked eye.Gels were destained in order to remove stain from areas of the gel whereno protein is located to make the stained areas contrast more vividlywith the transparent unstained background gel area. Destaining was doneby immersing the gels in several changes of acid alcohol until thebackgrounds were clear.

As shown in FIG. 5, two major differences between the banding patternsof TEPA and PEHA ampholytes are obvious. First, proteins which wereseparated in the PEHA gradient are more evenly distributed across thegradient and are therefore more easily resolved than those in the TEPAgel. Second, proteins analyzed by IEF in the TEPA gradient were spreadthrough the gel by obvious gaps (see arrow on FIG. 5) where no proteinwas detectable. FIG. 5 demonstrates that the fewer the number ofampholyte species, the greater the chance for artifactual grouping ofadjacent proteins, resulting in decreased resolution. This grouping iscaused when proteins migrating in an area of low buffering artifactuallymove to the closest available area of buffering. Thus, if there are nobuffering species of carrier ampholytes at, for instance, pH 5.2, butthere are carrier ampholyte species at pH 5.4, a protein with a pI of5.2 will move to the pH 5.4 area creating an artifact, or false result.Therefore, a relationship is established between the relativeheterogeneity of picric acid precipitable ampholyte species and thepotential resolution of proteins in ampholyte gradients.

e. Bis-Acrylamide CrossLinked Polyamines and IPG Precipitation

Picric acid has been previously reported to precipitate all commerciallyavailable ampholytes. In the search for synthetic methods to increaseampholyte diversity one method was found which did not produce picricacid precipitable ampholytes. This method involves the use ofbis-acrylamide to crosslink the polyamine backbone to create moreheterogeneous ampholytes. Picric acid analysis of bis-acrylamidecrosslinked ampholytes is shown in FIG. 6. Bis-acrylamide appears to bereacting with both TETA and TEPA in such a way that the resultingampholyte species are modified. This is clearly demonstrated by thereduction of the major picric acid precipitable species which areevident in the unmodified TETA and TEPA preparations. Under certainconditions (TETA:BIS ratio of 4:1 and TEPA:BIS ratio of 3:1) new speciesof picric acid precipitable ampholytes were prepared. As the ratio ofbis-acrylamide to TEPA increased, the number of picric acid precipitablespecies decreased.

This aspect of the invention demonstrates the application of IPGampholyte analysis for new ampholyte syntheses. Polyamine backbones suchas TETA or TEPA which produce poor ampholytes have been modified toproduce more diverse ampholyte species. The effect of thesemodifications have been monitored by IPG analysis. Detection of focusedampholyte species in the IPG is dependent upon the formation of avisible complex with picric acid. Picric acid combines with certainamino acids to form picrates and is used routinely in histologicalfixatives.

Not all ampholytes tested precipitated in picric acid. However, theampholyte species which did precipitate with picric acid appeared to bedistributed across the entire pH gradient, demonstrating thatprecipitation is not simply a function of the pI of the ampholytespecies. A direct correlation has been established between the numberand diversity of picric acid precipitable ampholyte species and thepotential resolution of focused proteins using these ampholytes inconventional IEF.

The IPG picric acid method of ampholyte analysis allows a qualitative orquantitative comparison of ampholytes which have been produced byvarious synthetic modifications. The analysis covers a continuous pHspectrum and is not limited by the availability of appropriate pI markerproteins to assess the stepless characteristics of the ampholyte. Thismethod of analysis insures that the ampholytes themselves are notdictating their own distribution patterns. The patterns are dictated bythe immobilized pH gradient which is independent of the ampholyte beinganalyzed. The development of ampholytes with such characteristics may beimportant in the analysis of protein mixtures such as polyclonalantibody preparations, which are vastly heterogeneous with respect totheir isoelectric points. Ampholytes which have many species within anarrow pH range may also be particularly attractive for preparative IEFapplications. The potential to expand narrow segments of the pHgradients for preparative IEF applications using narrow range amphoyletefractions isolated by recycling IEF would be more attractive if morespecies of carrier ampholyte per pH unit were available to separatebiomolecules in extremely narrow pH ranges.

Further modifications and alternative embodiments of various aspects ofthe invention will be apparent to those skilled in the art in view ofthis description. Accordingly, this description is to be construed asillustrative only and is for the purpose of teaching those skilled inthe art the general manner of carrying out the invention. It is to beunderstood that the forms of the invention shown and described hereinare to be taken as the presently preferred embodiments. Elements andmaterials may be substituted for those illustrated and described herein,parts and processes may be reversed, and certain features of theinvention may be utilized independently, all as would be apparent to oneskilled in the art after having the benefit of this description of theinvention. Changes may be made in the elements described herein or inthe steps or in the sequence of steps of the methods described hereinwithout departing from the spirit and scope of the invention asdescribed in the following claims. Similarly, isomers and homologs ofreactants may be used and still come within the scope of the invention.

What is claimed is:
 1. A method of analyzing chemical compounds,comprising the steps of:applying a chemical compound to an immobilizedpH gradient; isoelectrically focusing the chemical compound on theimmobilized pH gradient; applying a precipitating chemical solution tothe immobilized pH gradient and the isoelectrically focused chemicalcompound; precpitating the isoelectrically focused chemical compound;and visualizing the chemical compound.
 2. The method of claim 1, furthercomprising the step of visually comparing the visualized compound withat least one other visualized compound.
 3. The method of claim 2,further comprising the step of preparing at least one additionalcompound based on the visual comparisons.
 4. The method of claim 1,further comprising the step of agitating the immobilized pH gradient. 5.The method of claim 1, or 4, further comprising the step of prefocusingthe immobilized pH gradient.
 6. The method of claim 5, furthercomprising the step of hydrating the immobilized pH gradient prior toprefocusing.
 7. The method of claim 1, wherein the precipitatingchemical solution is saturated.
 8. The method of claim 1 wherein theprecipitating chemical solution is picric acid.
 9. The method of claim 1wherein the immobilized pH gradient comprises polyacrylamide.
 10. Themethod of claim 9 wherein the immobilized pH gradient comprises 1-10%acrylamide.
 11. The method of claim 9 wherein the immobilized pHgradient comprises 3-7% acrylamide.
 12. The method of claim 9 whereinthe immobilized pH gradient comprises 1-5% crosslinker.
 13. The methodof claim 9 wherein the immobilized pH gradient comprises 1-4%crosslinker.
 14. The method of claim 1 wherein the immobilized pHgradient comprises chemical compounds having the formula:

    CH.sub.2 ═CH--CO--NH--R,

where R is 2-acrylamido-2-methylpropane, 2-acrylamido glycolic acid,N-acryloyl glycine, 3-acrylamido propanoic acid, 4-acrylamido butyricacid, 2-morpholino ethylacrylamide, 3-morpholino propylacrylamide,N,N-dimethyl aminoethyl acrylamide, N,N-dimethyl aminopropylacrylamide,N,N-diethyaminopropylacrylamide, QAE-acrylamide, or mixtures thereof.15. The method of claim 1, wherein the chemical compound comprises anampholyte.
 16. The method of claim 15 wherein the ampholyte is preparedusing a polyamine.
 17. The method of claim 15 wherein the ampholyte isprepared using a peptide.
 18. The method of claim 15 wherein theampholyte comprises a reaction product of a polyamine and an acid. 19.The method of claim 15 wherein the ampholyte comprises a reactionproduct of a polyamine and an unsaturated acid.
 20. The method of claim15 wherein the ampholyte comprises the reaction product of a polyamineand an α,β-unsaturated carboxylic acid.
 21. The method of claim 16wherein the polyamine comprises a polyethylene polyamine.
 22. The methodof claim 21 wherein the polyethylene polyamine is DETA, TETA, TEPA,PEHA, or mixtures thereof.
 23. The method of claim 21 wherein thepolyethylene polyamine comprises DETA.
 24. The method of claim 21wherein the polyethylene polyamine comprises TETA.
 25. The method ofclaim 21 wherein the polyethylene polyamine comprises TEPA.
 26. Themethod of claim 21 wherein the polyethylene polyamine comprises PEHA.27. The method of claim 17 wherein the peptide comprises a chain of 3-20amino acids.
 28. The method of claim 27 wherein the amino acid chain isL-lysine, D-lysine, L-arginine, D-arginine, L-histidine, D-histidine, ormixtures thereof.
 29. The method of claim 15 wherein the ampholyte isthe reaction product of a crosslinked polyamine and an α,β-unsaturatedcarboxylic acid, and wherein the crosslinked polyamine is the reactionproduct of a polyamine and tartaric acid ester, malonic acid ester, or apolycarboxylic acid ester of the citric acid cycle.
 30. The method ofclaim 29 wherein the α,β-unsaturated carboxylic acid is acrylic acid,methacrylic acid, methylene malonic acid, ethylene malonic acid,crotonic acid, maleic acid, fumaric acid, or itaconic acid.
 31. Themethod of claim 29 wherein the α,β-unsaturated carboxylic acid comprisesacrylic acid.
 32. The method of claim 30 wherein the α,β-unsaturatedcarboxylic acid comprises methacrylic acid.
 33. The method of claim 29wherein the tartaric acid ester comprises L-diethyltartaric acid ester,D-diethyltartaric acid ester, meso diethyltartaric acid ester, racemicdiethyltartaric acid ester, or mixtures thereof.
 34. The method of claim29 wherein the polycarboxylic acid ester of the citric acid cycle iscitric acid ester, oxaloacetic acid ester, aconitic acid ester,oxalosuccinic acid ester, α-ketoglutaric acid ester, isocitric acidester, malic acid ester, or mixtures thereof.
 35. The method of claim 1,further comprising the step of photographing the precipitated chemicalcompounds.
 36. The method of claim 1, wherein the chemical compoundcomprises a mixture of amphoteric substances.
 37. The method of claim 1,further comprising the step of quantitative measurement of theprecipitated chemical compound by densitometric scanning.
 38. The methodof claim 1 wherein the chemical compound is precipitated into regions ofseparate species.
 39. The method of claim 38 wherein the precipitatedregions are measured by densitometric scanning.
 40. The method of claim37 wherein the densitometric scanning quantitates the amount of eachprecipitated region as a percentage of the whole chemical compound.